1. Field of Invention
This invention relates to bacteria and enzymes effective for hydrolyzing alternan to lower molecular weight fragments having reduced viscosity.
2. Description of the Prior Art
The polysaccharide alternan was first described by Jeanes et al. (1954, J. Am. Chem. Soc., 76:5041-5052) as one of two extracellular .alpha.-D glucans, referred to as fraction S, produced by Leuconostoc mesenteroides NRRL B-1355. The structure of this fraction was later determined by Misaki et al. (1980, Carbohydr. Res., 84:273-285) to consist primarily of an alternating sequence of .alpha.-1,3- linked and .alpha.-1,6- linked D-glucose residues, with approximately 10% branching. Because the .alpha.-1,3- linkages are part of the linear chain of the S fraction and there are not any consecutive .alpha.-1,6-linkages, the fraction is not a true dextran, and Cote and Robyt (1982, Carbohydr. Res., 101:57-74) therefore named this fraction alternan. These authors also isolated the enzyme alternansucrase which synthesizes alternan from sucrose.
Native, high-molecular weight alternan may be produced fermentatively as described by Jeanes et al. (1954, ibid) or enzymatically as described by Cote and Robyt (1982, ibid). This compound, its low molecular weight derivatives produced by sonication, and limit alternan produced by hydrolysis with the isomalto (G.sub.2)-dextranase of Arthrobacter globiformis T6 (Misaki et al., 1980, ibid), have properties resembling certain functional characteristics of gum arabic, maltodextrins, or Polydextrose (Cote, 1992, Carbohydrate Polymers, 19:249-252). The low viscosities of these products lend themselves to potential commercial applications as substitutes for gum arabic, for use as bulking agents and extenders in foods and cosmetics, particularly as noncaloric, carbohydrate-based soluble food additives in artificially sweetened foods.
Although alternan is an .alpha.-D-glucan, no endo-hydrolytic enzymes have been described which are capable of hydrolyzing alternan to any great extent (Cote, 1992, ibid). Misaki et al. (1980, ibid) reported that an endo-dextranase from a Penicillium species hydrolyzed alternan to a small degree, 7.3%. However, in subsequent work, Cote and Robyt (1982, ibid) examined the effect of this and other endo-dextranases upon alternan and found that the enzymes were unable to hydrolyze alternan, and no measurable low molecular weight products were produced. This lack of activity was not surprising, because the endo-dextranases are specific for dextrans, requiring consecutively linked .alpha.-1,6-D-glucose residues as substrates, while alternan is composed of alternating .alpha.-1,3- and .alpha.-1,6-linkages.
In contrast to what one may expect, alternan is considerably resistant to microbial degradation and is also not attacked by enzymes that degrade starch, nigeran or pullulan (Cote, 1992, ibid). The only enzymes that have been reported to hydrolyze alternan to any significant extent are isomaltodextranases, which are not endo-hydrolases but rather exo-hydrolases or exo-dextranases. Two isomaltodextranases were examined for hydrolysis of alternan (referred to as B-1335 fraction S), the isomaltodextranases produced by Arthrobacter globiformis (Sawai et al., 1978, Carbohydrate Res., 66:195-205) and by an actinomycete Actinomadura (Sawai et al., 1981, Carbohydrate Res., 89:289-299). The authors concluded that the isomaltodextranases release mainly isomaltose units from the non-reducing ends of alternan chains that are terminated with an .alpha.-1,6-linked D-glucopyranosyl residues.
Although there is firmer evidence for exo-action of the Actinomadora isomaltodextranase (Sawai et al., 1981, ibid), the information on the mode of action of the A. globiformis enzyme is not so straightforward. Its alternan digest also contained, besides isomaltose, some larger oligosaccharide fragments, identified by Sawai et al. (1981, ibid) as .alpha.-D-Glcp-(1,6)-.alpha.-D-Glcp-(1,3)-.alpha.-D-Glcp-(1,6)-.alpha.-D-G lc, and .alpha.-D-Glcp-(1,3)-.alpha.-D-Glcp-(1,6)-.alpha.-D-Glcp-(1,3)-.alpha.-D-G lcp-(1,6)-D-Glc. The authors suggested that these larger products represented either fragments that originated in the reducing-end terminals remaining after an exo-pattern of alternan digestion or, they were transisomaltosylation products. The second alternative finds support in the fact that isomaltodextranase is a glycanase retaining the anomeric configuration of attacked linkages in the hydrolysis products (Sawai & Niwa, 1975, Agr. Biol. Chem., 39:1077-1083). The A. globiformis isomaltodextranase has also been shown to cleave all types of .alpha.-glucopyranosyl linkages following an isomaltosyl unit towards the reducing end of the substrate. In other words, the enzyme liberates terminally linked isomaltose whether the linkage is .alpha.-1,6-, .alpha.-1,4-, .alpha.-1,3- or .alpha.-1,2-(Torii et al., 1976, Biochem. Biophys. Res. Comm., 70:459-464).
The recent observation by Okada et al. (1988, Agr. Biol. Chem, 52:829-836) that A. globiformis isomaltodextranase is capable of attacking pullulan in an endo-fashion cleaving the .alpha.-1,4-linkages following the .alpha.-1,6-linkage, raised again the question concerning the exo- or endo-character of the enzyme. However, studies with A. globiformis isomaltodextranase purified in this laboratory according to Okada et al. (1988, Agric. Biol. Chem., 52:495-501) have confirmed that the enzyme is not capable of endo-hydrolytic cleavage of alternan, but functions in an exo- fashion.